Carbonic anhydrase activators: Activation of the β-carbonic anhydrase Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids

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Carbonic anhydrase activators: Activation of the b-carbonic anhydrase

Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids

Semra Isik

a

, Feray Kockar

b

, Meltem Aydin

b

, Oktay Arslan

a

, Ozen Ozensoy Guler

a

, Alessio Innocenti

c

,

Andrea Scozzafava

c

_{, Claudiu T. Supuran}

_c,*

a

Balikesir University, Science and Art Faculty, Department of Chemistry, Balikesir, Turkey

b

Balikesir University, Science and Art Faculty, Department of Biology, Balikesir, Turkey

c

Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy

a r t i c l e

i n f o

Article history:

Received 26 December 2008 Revised 30 January 2009 Accepted 30 January 2009 Available online 5 February 2009 Keywords: Carbonic anhydrase Beta-class enzyme Saccharomyces cerevisiae Candida albicans Cryptococcus neoformans Amine Amino acid Enzyme mechanism

a b s t r a c t

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a b-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such asL-/D-His, Phe, DOPA, Trp (KAs of 82–90lM) and more effectively

activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-pipera-zine/morpholine (KAs of 10.2–21.3lM). The best activator wasL-adrenaline, with an activation constant of 0.95lM. This study may help to better understand the catalytic/activation mechanisms of the b-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.

Modulation of the enzyme activity of carbonic anhydrases (CAs, EC 4.2.1.1) represents a means of therapeutic intervention1–3for a variety of diseases due to the fact that these metalloenzymes are involved in critical physiologic processes in organisms all over the phylogenetic tree, from bacteria and archaea to plants, fungi, and animals.1–5 _{Indeed, there are ﬁve independently-evolved}

(

a

, b,

c

, d, and f) classes of CAs reported up to date, of which the

a

-class from mammalian sources has been studied to a far greater extent than the other four classes.1–3_{Yet, CAs other than those}

belonging to the

a

-class are widely distributed in nature, with the b-CAs being the most abundant such catalysts for the intercon-version between carbon dioxide and the bicarbonate ions.1,2,5

Re-cent work has shown that various CAs are widespread in metabolically diverse species from both the Archaea and Bacteria but also in microscopic eukaryotes, such as yeast or pathogenic fungi, indicating that these enzymes have a more extensive and fundamental role than originally recognized.1,6,7

Whereas inhibition of CAs was investigated in great detail (again, mainly for the

a

-CAs from mammals)1,3,8,9 _{with several}

CA inhibitors (CAIs) in clinical use as diuretics, antiglaucoma,

anti-obesity or anticancer agents/diagnostic tools,1,3,8 _{CA activators}

(CAAs) received less attention and only in the last 10 years this class of enzyme modulators started to be investigated systemati-cally for their interaction with mammalian

a

-CAs.10 _{Indeed, our}

group reported several kinetic and X-ray crystallographic studies regarding the interaction of all mammalian isoforms (CA I–XIV) with amino acid and amine types of activators, also unraveling the activation mechanism of these enzymes.10–14

The rate-determining step in the CA catalytic cycle for CO2

hydration to bicarbonate is the formation of the zinc hydroxide species of the enzyme.1–5 _{This involves the transfer of a proton}

from a Zn(II)-coordinated water molecule to the environment, which can be assisted by amino acid residues from the enzyme ac-tive site (such as His64 in CA II, IV, VII, IX, XII, XIII, and XIV)10_{or by}

an activator molecule bound within the cavity.10–14Such phenom-ena are now well understood for the

a

-CAs (with many X-ray crys-tal structures of enzyme-activator adducts available)10–14 _but

started to be investigated only recently for enzymes belonging to the b- and

c

-classes. Indeed, recently a ﬁrst activation study of the b- and

c

-CAs from some Archaea was reported by this group.15

The activation proﬁle of the b-CA from Methanobacterium thermo-autotrophicum (Cab) and the

c

-class enzyme from Methanosarcina thermophila (Cam) with a series of amine and amino acids is very

*Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573835. E-mail address:claudiu.supuran@uniﬁ.it(C.T. Supuran).

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1662–1665

Contents lists available atScienceDirect

Bioorganic & Medicinal Chemistry Letters

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m c l

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different as compared to those of the mammalian

a

-CAs,15 _but

seems to operate via the same mechanism of action, that is, the activator bound within the enzyme active site facilitates the shut-tling of protons between the Zn(II) ion-coordinated water molecule and the environment, with generation of the nucleophilic zinc hydroxide, catalytically active species of the enzymes.15

Recently we have cloned and characterized kinetically a b-CA encoded by the Nce103 gene of the yeast Saccharomyces cerevisiae, denominated scCA.16_{Its inhibition with inorganic}

metal-complex-ing anions and sulfonamides has also been investigated.16_Indeed,

S. cerevisiae, one of the most studied budding yeasts and a widely used model of eukaryotic organisms has a genome comprising 6275 genes condensed into 16 chromosomes, which was com-pletely sequenced in 1996.17_{The gene Nce103 (from non-classical}

export), was originally reported by Cleves et al. to encode for a pro-tein involved in a non-classical propro-tein secretion pathway.18 Sub-sequently, it has been shown by several groups that this protein is a b-CA required to provide sufﬁcient bicarbonate for essential metabolic carboxylation reactions of the yeast metabolism, such as those catalyzed by pyruvate carboxylase (PC), acetyl-CoA car-boxylase (ACC), carbamoyl phosphate synthase (CPSase) and phos-phoribosylaminoimidazole (AIR) carboxylase.19,20

N H N NH₂ OH OH NH₂ N H NH₂ HO N NH₂ _X _N NH2 H₂N _H 2N H₂N H₂N H2N H2N O N N OH O OH O OH OH OH O N OH O OH OH O NH2 OH HO OH N H OH H 11 12 13 ( )n 14: n = 1 15: n = 2 16: X = NH17: X = O 1: L-His

2: D-His 3: L-Phe4: D-Phe 5: L-DOPA6: D-DOPA

7: L-Trp 8: D-Trp

9: L-Tyr 10: 4-H2N-L-Phe

18

Here we report the ﬁrst activation study of scCA with a series of amines and amino acids (of types 1–18), investigated earlier10–14

for their interaction with mammalian

a

-CAs as well as very re-cently15with the b- and

c

-class enzymes from the Archaea domain. Such a study may help a better understanding of the b-CA catalytic/ activation mechanism (the natural proton shuttling residue in this class of enzymes has not been yet identiﬁed), as well as the design of CAAs targeting other b-CAs from pathogenic organisms, such as the closely related enzymes from Candida albicans (Nce103, that is, encoded by the orthologue gene of S. cerevisiae present in the

path-ogenic fungus) and Cryptococcus neoformans (Can2).21_{The cloning,}

kinetic characterization and inhibition with anions and sulfona-mides of these enzymes present in pathogenic fungi were recently reported by this group.21

scCA has been overexpressed16_{in Escherichia coli and puriﬁed}

by an original procedure leading to high amounts of pure protein possessing a good enzyme activity for the physiological reaction, that is, CO2 hydration to bicarbonate. Similarly to other CAs

belonging to the

a

- or b-class, the yeast CAs enzyme scCA pos-sesses appreciable CO2 hydrase activity, with a kcat of

9.4 105_s1_{, and k}

cat/Kmof 9.8 107M1s1.16Data ofTable 122

show that histamine, Hsn (at 10

l

M concentration), which is an effective CAA for scCA (see later in the text) and a less effective one for Cab and hCA II,15_{enhances k}

catvalues for all these enzymes,

whereas KMremains unchanged. Hsn is a micromolar activator for

the

a

-class enzyme (hCA II), with KAof 125

l

M,11–14being a more

effective micromolar one for the archaeal one Cab (KAof 76

l

M)

and yeast enzyme scCA investigated here (KAof 20.4

l

M, see

dis-cussion later in the text). It is thus obvious that the activation mechanism of the

a

- and b-CAs seems to be similar, that is, the activator enhances kcatwith no inﬂuence on KM, facilitating thus

the release of the proton from water coordinated to the catalytic zinc ion.

Data ofTable 2 show that all amino acids and amines 1–18 investigated here act as CAAa against the yeast enzyme scCA, but with very different potencies (activation of the

a

-class enzymes hCA II and the b-one Cab, investigated earlier10–15_{are included in} Table 2for comparison reasons). The following structure activity relationship (SAR) can be observed for the activation of these CAs with compounds 1–18:

(i) scCA23_{was activated rather inefﬁciently by amino acids 1–9,}

which showed activation constants22 _{in the range of 82–}

90

l

M. It may be observed that all these aromatic/heterocy-clic amino acids show a rather ﬂat SAR, being weak scCA activators, whereas some of them are much more effective Cab (D-Pe,L-DOPA,D-DOPA andL-Trp) or hCA II (L- and D

-Phe,L- andD-DOPA,L-Tyr) activators (Table 2). The

enantio-meric form (L- orD-) as well as the substitution pattern (in b to the carboxyl group) of these amino acid derivative were non-inﬂuential to their activating efﬁcacy.

(ii) A second group of derivatives, including 4-amino-phenylal-anine 10, amines 11–15 and 17, showed more effective scCA activating power as compared to the previously discussed compounds, with KAs in the range of 10.2–21.3

l

M. The

main difference between amino acid 10 and derivatives 1– 9 mentioned above, is the presence of the supplementary

Table 1

Kinetic parameters for the activation of human (hCA) isozyme II, Cab and scCA with histamine (Hst), measured at 25 °C, pH 8.3 in 20 mM Tris buffer and 20 mM NaClO4,

for the CO2hydration reaction22

Isozyme kcat*(s1) KM*(mM) (kcat)Hst**(s1) KA***(lM) Hst

hCA IIa _{1.4 10}6 _9.3 _{2.0 10}6 ₁₂₅ Cabb 3.1 104 1.7 4.5 104 76 scCAc 9.4 105 9.5 19.6 105 20.4

*_{Observed catalytic rate without activator. K}

Mvalues in the presence and the

absence of activators were the same for the various CAs (data not shown).

** _{Observed catalytic rate in the presence of 10}_l_{M activator.} *** _{The activation constant (K}

A) for each enzyme was obtained by ﬁtting the

observed catalytic enhancements as a function of the activator concentration.22

Mean from at least three determinations by a stopped-ﬂow, CO2hydrase method.22

Standard errors were in the range of 5–10% of the reported values.

a

Human recombinant enzyme, data from Ref.10.

b

Archaeal recombinant enzyme, data from Ref.15.

c

Yeast recombinant enzyme.

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amino moiety in 10, attached to the aromatic ring, whereas derivatives 1–9 possess only the aliphatic amine moiety. On the other hand, histamine 11 is quite structurally similar to 1 and 2, from which it can be formed by a decarboxylation reaction. The same is true for dopamine 12 andL-/D-DOPA 5 and 6. It may be observed that the biogenic amines 11 and 12 are around 4.1–6.9 times more effective scCA activa-tors as compared to the structurally related amino acids 1/2 and 5/6, respectively. The pyridyl-alkylamino derivatives 14 and 15 are also effective scCA activators, with the amino-ethyl derivative 15 being slightly more effective than the aminomethyl one 14. It is thus clear that minor structural variations in the scaffold of an amine/amino acid, strongly inﬂuence their interaction with scCA active site and as a con-sequence, their activation properties. It is also obvious from data ofTable 2that the activation proﬁles of hCA II and Cab with these compounds is quite different from those of scCA. Generally amines 10–18 act as effective scCA activators being less effective as hCA II or Cab activators. The reverse is true for many amino acid derivatives 1–9, as mentioned above.

(iii) The most potent scCA activators were the piperazine deriv-ative 16 (KAof 9.3

l

M) andL-adrenaline 18, the only

com-pound showing a submicromolar activation constant (KAof

0.95

l

M). Whereas 16 is a medium potency activator of Cab and an efﬁcient hCA II activator,L-adrenaline 18 shows

very weak hCA II activating properties (KAof 96

l

M) being

a more effective Cab activator. The weak hCA II activating properties of L-adrenaline were explained by us after the

report of the X-ray crystal structure of the hCA II–18 adduct,14a in which the activator was seen bound in an unexpected region of the active site, plugging the entrance to it, and being unable to favorably shuttle protons, unlike histamine,10bL-/D-His13corL-/D-Phe,11ewhich bind in differ-ent regions of the CA II active site and actively participate to the transfer of protons between the active site and the envi-ronment. Thus,L-adrenaline may be considered a potent and

also rather selective scCA activator. At this moment it is

unclear whether activation of scCA with this type of com-pounds may have physiological relevance but studies in this ﬁeld are clearly warranted. Considering the fact that scCA is involved in carboxylation reactions of the yeast metabo-lism,19,20_{it is possible that its activation may have relevant}

consequences for the growth of S. cerevisiae and might be exploited biotechnologically.

A possible activation mechanism of the b-CAs is depicted sche-matically inFigure 1. As for other fungal b-CAs, the catalytic Zn(II) ion in the scCA active site is coordinated to residues Cys106, His161 and Cys164 (Nce103 of C. albicans numbering system).16,21

A second pair of conserved amino acid residues in all sequenced b-CAs known to date,2,16,21 _{is constituted by the dyad Asp108–}

Arg110 (Nce103 of C. albicans numbering, Fig. 1). These amino acids are close21to the zinc-bound water molecule, which is the fourth Zn(II) ligand in this type of open active site b-CAs,21

partic-ipating in a network of hydrogen bonds with it, which probably as-sist water deprotonation and formation of the nucleophilic, zinc hydroxide species of the enzyme. The active site channel of b-CAs (as exempliﬁed by the recently determined X-ray crystal struc-ture of the C. neoformans enzyme Can2)21b_{is a channel which can}

accommodate elongated molecules such as the aromatic amino acids/amines investigated here. Thus, we hypothesize that the acti-vators bind nearby the pocket deﬁned by Asp108/Arg110, estab-lishing supplementary hydrogen bonds with the polar moieties of these amino acids or with the zinc-bound water molecule (directly or through a relay of several other water molecules, as demon-strated for the interaction of

a

-CAs with this type of activator)10–

14_{assisting thus water deprotonation and facilitating the catalytic}

turnover. Indeed, both the amino or carboxyl moieties of these activators can establish hydrogen bonds with these structural ele-ments, due to the presence of many heteroatoms in their mole-cules.Figure 1shows schematically a putative binding mode ofL

-adrenaline 18 within the active site of scCA. This hypothesis should be checked by X-ray crystallography, but the structure of scCA is not yet reported.

In conclusion, we report the ﬁrst activation study of the b-CA from the yeast S. cerevisiae with amines and amino acids. scCA was poorly activated by amino acids such asL-/D-His, Phe, DOPA,

Trp (KAs of 82–90

l

M) and more effectively activated by amines

such as histamine, dopamine, serotonin, pyridyl-alkylamines, ami-noethyl-piperazine/morholine (KAs of 10.2–21.3

l

M). The best

activator was L-adrenaline, with an activation constant of Table 2

Activation constants of hCA II (cytosolica-isozyme), Cab (archaeal CA) and yeast b-CA from S. cerevisiae (scb-CA) with amino acids and amines 1–18. Data for hb-CA II and Cab activation with these compounds are from Ref.15

No. Compound KA(lM)*

hCA IIa _Cabb _scCAc

1 L-His 10.9 69 82 2 D-His 43 57 85 3 L-Phe 0.013 70 86 4 D-Phe 0.035 10.3 86 5 L-DOPA 11.4 11.4 90 6 D-DOPA 7.8 15.6 89 7 L-Trp 27 16.9 91 8 D-Trp 12 41 90 9 L-Tyr 0.011 10.5 85 10 4-H2N-L-Phe 0.15 89 21.3 11 Histamine 125 76 20.4 12 Dopamine 9.2 51 13.1 13 Serotonin 50 62 15.0 14 2-Pyridyl-methylamine 34 18.7 16.2 15 2-(2-Aminoethyl)pyridine 15 40 11.2 16 1-(2-Aminoethyl)-piperazine 2.3 13.8 9.3 17 4-(2-Aminoethyl)-morpholine 0.19 18.5 10.2 18 L-Adrenaline 96 11.5 0.95

*_{Mean from three determinations by a stopped-ﬂow, CO}

2hydrase method.22

Standard errors were in the range of 5–10% of the reported values.

a_{Human recombinant isozyme, from Ref.}₁₅_. b _{Recombinant archaeal enzyme, from Ref.}₁₅_. c

Recombinant yeast enzyme, this study.23,24

OH OH N H HO H O O N H N M O S S H H N H H₂N NH His161 Cys164 Cys106 2+ -Asp108 -Arg110

Figure 1. Proposed schematic interactions between an activator (L-adrenaline 18) and the scCA active site.

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0.95

l

M. This study may help to better understand the catalytic/ activation mechanisms of the b-CAs and eventually to design mod-ulators of CA activity for similar enzymes present in pathogenic fungi, such as C. albicans and C. neoformans.

Acknowledgments

This research was ﬁnanced in part by a grant of the 6th Frame-work Programme of the European Union (DeZnIT project), to A.S. and C.T.S.

References and notes

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19. (a) Götz, R.; Gnann, A.; Zimmermann, F. K. Yeast 1999, 15, 855; (b) Amoroso, G.; Morell-Avrahov, L.; Muller, D.; Klug, K.; Sultemeyer, D. Mol. Microbiol. 2005, 56, 549.

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22. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-ﬂow instrument was used for assaying the CA catalyzed CO2hydration activity.

Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, 10–20 mM Hepes (pH 7.5) or Tris (pH 8.3) as buffers, and 20 mM Na2SO4or 20 mM NaClO4(for maintaining constant the

ionic strength), following the CA-catalyzed CO2hydration reaction for a period

of 10 s at 25 °C. The CO2concentrations ranged from 1.7 to 17 mM for the

determination of the kinetic parameters and activation constants. For each activator at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activators 1–18 (10 mM) were prepared in distilled-deionized water and dilutions up to 0.001lM were done thereafter with distilled-deionized water. Activator and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–A complex. The activation constant (KA), deﬁned similarly with the inhibition

constant KI,1–3can be obtained by considering the classical Michaelis–Menten

equation (Eq.1), which has been ﬁtted by non-linear least squares by using PRISM 3:

v

¼

v

max=f1 þ KM=½Sð1 þ ½Af=KAÞg ð1Þ where [A]fis the free concentration of activator.

Working at substrate concentrations considerably lower than KM([S] KM),

and considering that [A]fcan be represented in the form of the total

concentra-tion of the enzyme ([E]t) and activator ([A]t), the obtained competitive

steady-state equation for determining the activation constant is given by Eq.2:10–14

v

¼

v

0:KA=fKAþ ð½At 0:5fð½Atþ ½Etþ KAÞ ð½At

þ ½Etþ KAÞ2 4½At½EtÞ 1=2

gg ð2Þ

wherev0represents the initial velocity of the enzyme-catalyzed reaction in the

absence of activator.10–14

23. Yeast genomic DNA was isolated using the Johnston’s procedure (Johnston, J. R. Molecular Genetics of Yeast; Oxford University Press: New York, 1994). The Nce103 gene was ampliﬁed from genomic DNA by PCR based strategies using the following oligonucleotides; NCE103ORF-for (50_{-AGGATCCATGAGCG}

CTACCGAA-30₎ _and _{NCE103ORF-rev} ₍₅0_{-AGAGCTCCTATTTTGGGGTAAC-3}0_).

PCR conditions were: 94 °C for 2 min, 35 cycles of 94 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min and a ﬁnal step of 72 °C for 10 min. The ampliﬁed band containing Nce103 ORF was inserted into the pGEM-T (PROMEGA) vector with T:A strategy.24

Automated sequencing of the clone was performed in order to confirm the gene and the integrity of amplified gene. The construct was then excised with BamH I and Sac I restriction enzymes and subcloned into pET21a(+) expression vector. The vectors were transformed into E. coli BL21 (DE3) competent cells. The enzyme was purified as reported earlier16

. 24. Promega Technical Manual ‘pGEM-T and pGEM-T Easy Vector Systems’,

available atwww.promega.com.